Bacillus Velez L09 strain from crocodile and its application in aquaculture

Abstract

The present invention provides a crocodile-derived Bacillus velezensis L09 strain and its application in aquaculture. The crocodile-derived Bacillus velezensis L09 strain has been deposited in the China Center for Type Culture Collection (CCTCC) on July 31, 2024, with a deposit address of Wuhan University, Wuhan, China, a deposit number of CCTCC NO: M 20241703, and a classification name of Bacillus velezensis. The crocodile-derived Bacillus velezensis L09 strain has in vivo and in vitro antibacterial activity against zebrafish infected with Aeromonas dakar, and has good application prospects in aquaculture.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to a crocodile-derived Bacillus Velez subtilis L09 strain and application thereof in aquaculture. Background Art

[0002] The homeostasis of intestinal microorganisms plays a key regulatory role in preventing animal diseases and maintaining health. Intestinal microorganisms can also accelerate the normal development of the gastrointestinal tract and promote the digestion and absorption of nutrients. This is because some carbohydrates that cannot be consumed and absorbed in the small intestine are produced by intestinal microorganisms under anaerobic fermentation to produce short-chain fatty acids (SCFA), mainly including acetate, propionate and butyrate, providing 10% of their daily requirement. The production of some of these SCFAs is beneficial to the host, affecting the host's immune and metabolic systems, improving intestinal barrier function, reducing intestinal inflammation, and even affecting the host's brain function.

[0003] Bacillus is a probiotic that can be used to remove mycotoxins and has great potential as a feed additive. Bacillus promotes intestinal health by producing antimicrobial peptides to inhibit the toxicity of bacterial pathogens. It can also promote the production of beneficial metabolites by intestinal flora, regulate intestinal health, and even play an important role in improving the human immune system and stimulating body growth.

[0004] Bacillus velezensis is an aerobic, Gram-positive, endospore-forming bacterium that promotes plant growth. Many strains of this species have been reported to inhibit the growth of microbial pathogens, including bacteria, fungi, and nematodes. Based on a recent phylogenetic analysis, several Bacillus species have been reclassified as B. velezensis. Genomic analysis revealed that B. velezensis possesses strain-specific gene clusters associated with the biosynthesis of secondary metabolites, which play important roles in pathogen suppression and plant growth promotion. However, there are very few reports on B. velezensis isolated from fish and the ability of this bacterium to antagonize pathogens in vivo. Summary of the invention

[0005] Based on the above, the purpose of the present invention is to provide a crocodile-derived Bacillus Velez subtilis L09 strain and its application in aquaculture, and to isolate a Bacillus Velez subtilis that can antagonize the important pathogenic bacteria Aeromonas dakar in water from crocodile feces, which can be used in aquaculture.

[0006] The technical solution adopted by the present invention is:

[0007] The present invention provides a crocodile-derived Bacillus velezensis L09 strain, which was deposited in the China Center for Type Culture Collection (CCTCC) on July 31, 2024, with a deposit address of Wuhan University, Wuhan, China, a deposit number of CCTCC NO: M20241703, and a classification name of Bacillus velezensis.

[0008] Furthermore, the total genome length of the crocodile-derived Bacillus Velez subtilis L09 strain is 3810733 bp, and the average GC content is 46.69%.

[0009] Furthermore, the genome of the crocodile-derived Bacillus Velez subtilis L09 strain contains a cluster of stimulatory metabolites related to antibacterial substances, including polyketide compounds, lipopeptide compounds, and bacillusin.

[0010] Furthermore, the whole genome of the crocodile-derived Bacillus Velez strain L09 contains five families of CAZymes: carbohydrate binding modules, carbohydrate lipolytic enzymes, glycoside hydrolases, glycosyltransferases and polysaccharide lyases.

[0011] Furthermore, the crocodile-derived Bacillus Velezii L09 strain is sensitive to the following antibiotics, including neomycin, nitrofurantoin, streptomycin, chloramphenicol, fosfomycin, tobramycin, roxithromycin, cefazolin, cephalexin, cefoperazone, polymyxin B, cefoxitin, cephradine, ciprofloxacin, sulfamethoxazole, minocycline, lincomycin, co-trimoxazole, clindamycin, spectinomycin, and norfloxacin.

[0012] The present invention also provides an aquatic antibacterial agent, comprising the crocodile-derived Bacillus Velez subtilis L09 strain.

[0013] The present invention further provides application of the crocodile-derived Bacillus Velez subtilis L09 strain in aquaculture.

[0014] Furthermore, the application includes the application of the crocodile-derived Bacillus Velez subtilis L09 strain as an antagonist against fish pathogenic bacteria.

[0015] Furthermore, the fish pathogenic bacteria include Aeromonas dhaka.

[0016] Furthermore, the fish pathogenic bacteria is Aeromonas dakar C160501.

[0017] The beneficial effects of the present invention are:

[0018] The present invention isolates a strain of Bacillus Velezii L09 that can antagonize Aeromonas dakar, an important pathogen in water, from crocodile feces in a crocodile farm in Dongfang City, Hainan Province, and has good effects. It is further verified that the strain of Bacillus Velezii L09 has in vivo and in vitro antibacterial activity against zebrafish infected with Aeromonas dakar, and reduces the mortality rate of tilapia infected with Aeromonas dakar. The main antagonistic mechanism seems to be to inhibit the growth of Aeromonas dakar by depriving the pathogen of nutrition or by secreting extracellular products and reducing its invasion and infection ability. It has good application prospects in aquaculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the growth curve of Bacillus Velezii L09.

[0020] Figure 2 This is the hemolytic activity test result of Bacillus Velezii L09 strain.

[0021] Figure 3 This is the 16S phylogenetic tree of Bacillus Velezii L09 strain based on genomic data.

[0022] Figure 4 This is the complete genome map of Bacillus Velez strain L09.

[0023] Figure 5 This is the whole genome variation map of Bacillus velez L09 strain.

[0024] Figure 6 The results of the detection of antagonistic bacterial metabolites in the genome of Bacillus Velezii L09 strain.

[0025] Figure 7 The results of CAZymes analysis in the genome of Bacillus velez L09 strain.

[0026] Figure 8 The results of in vitro co-culture of Bacillus Velez L09 against the growth of Aeromonas dakar C160501, A. Morphology of Bacillus Velez L09; B. Morphology of Aeromonas dakar C160501; C. Biomass of C160501 after 12h of co-culture of L09 and C160501; D. Biomass of C160501 after 12h of co-culture of L09 and C160501 (enlarged view of Figure C); E. Biomass of C160501 after 24h of co-culture of L09 and C160501; F. Biomass of C160501 after 48h of co-culture of L09 and C160501.

[0027] Fig. 9These are the results of the in vivo gavage test on zebrafish, A. PBS group; B. Aeromonas dakar C160501 group; C. CFE group; D. CFE and Aeromonas dakar C1605010 group; E. Bacillus velez L09 group; F. L09 and C160501 group; G. CFS group; H. CFS and C160501 group; the black box indicates the magnified range.

[0028] Fig.10 These are the 14-day survival results of the in vivo gavage experiment on zebrafish; Da: Aeromonas dasyphylla C160501 group; CFS-Da: extracellular secretions and C160501 group; CFE-Da: intracellular extracts and C160501 group; Ba-Da: Bacillus velezensis L09 and C160501 group; PBS: PBS group. DETAILED DESCRIPTION

[0029] In order to explain the present invention more clearly, the present invention is further described in detail below in conjunction with embodiments and with reference to the accompanying drawings. It should be understood by those skilled in the art that the content described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0030] The Bacillus velezensis L09 strain was deposited in the China Center for Type Culture Collection (CCTCC) on July 31, 2024. The deposit address is Wuhan University, Wuhan, China. The deposit number is CCTCC NO:M 20241703, and the classification name is Bacillus velezensis.

[0031] Because it comes from crocodile feces, it is also called crocodile-derived Bacillus Velez L09 strain.

[0032] Example

[0033] The present application aims to evaluate the in vivo and in vitro antibacterial activity of Bacillus velez against zebrafish infected with Aeromonas dakar, and whether it can promote intestinal homeostasis and protect intestinal health in zebrafish, a model animal.

[0034] 1. Isolation and identification of Bacillus velezinsis L09 strain

[0035] 50g of crocodile feces sample collected from a farm in Dongfang Crocodile Town, Dongfang City, Hainan Province, China was added to a flask containing 200mL LB and shaken on a constant temperature shaker at 180r / min for 2h to prepare a bacterial suspension. 2mL of bacterial suspension was added to a flask containing 100mL LB medium and incubated at 37℃ at 180r / min for 2d. 1mL of enriched culture medium was taken into a test tube and diluted 10 times in a gradient. -3 , 10 -4The bacterial suspension was placed in an 85°C water bath for 10 min. 100 μL of the bacterial suspension after the water bath was spread on LB solid medium and incubated at 37°C for 24 h. Different single colonies with obvious morphological characteristics were selected and streaked 2-3 times using the plate streak method to purify single colonies.

[0036] The growth curve of Bacillus velez Figure 1 It can be clearly observed that after 24 hours of cultivation, Bacillus Velezii still has a strong growth activity and maintains a steady upward state overall.

[0037] 2. Hemolytic Activity

[0038] Triplicate aliquots (5 μL) of the purified Bacillus Velez strain were incubated on Columbia agar plates containing 5% sterile defibrinated sheep blood. The diameter and color of the halo indicating hemolytic activity were then recorded, with the presence of clear transparent zones around the colonies (β-hemolysis) indicating positive hemolytic activity, and the presence of green zones (α-hemolysis) or the absence of clear zones (γ-hemolysis) indicating negative hemolytic activity. Each potential probiotic strain was tested twice using three replicates.

[0039] Results Figure 2 , a green area can be clearly observed on the hemolytic plate, indicating that the hemolytic activity is negative and it is a safe strain.

[0040] 3. Drug sensitivity test

[0041] Kirby-Bauer disk diffusion method was used to evaluate the results of susceptibility test of Bacillus velezensis L09 to 21 drugs. These included neomycin, nitrofurantoin, streptomycin, chloramphenicol, fosfomycin, tobramycin, roxithromycin, cefazolin, cephalexin, cefoperazone, polymyxin B, cefoxitin, cephradine, ciprofloxacin, sulfisoxazole, minocycline, lincomycin, cotrimoxazole, clindamycin, spectinomycin, and norfloxacin. In brief, bacterial suspension (200 μL) was cultured on LB or MRS solid medium (Hangzhou Microbiological Reagent Co., Ltd., Hangzhou, China) with antibiotic disks for 24 h. Bacillus velezensis was tested twice using three replicates. The results are shown in Table 1.

[0042] Table 1. Analysis of drug sensitivity test results

[0043]

[0044]

[0045] Note: R is resistant; I is moderately sensitive; S is sensitive

[0046] As shown in Table 1, the isolated and identified Bacillus Velez subtilis was sensitive to 21 antibiotics, which can ensure its good safety in the later feed preparation.

[0047] 4. Bacterial Framework and Resequencing

[0048] Genomic DNA was isolated from fresh bacterial cultures using a DNA extraction kit and quantified using a Nanodrop spectrophotometer.

[0049] Bacteria framework diagram: After the DNA samples are received, they are tested, and then the qualified samples are used to construct the library: first, the DNA samples are randomly interrupted to generate DNA fragments of the required length, the sticky ends formed by the interruption are repaired into flat ends, and then the base "A" is added to the 3' end so that the DNA fragments can be connected to the special adapter with the "T" base at the 3' end; finally, the PCR technology is used to amplify the DNA fragments with adapters at both ends, thus completing the construction of the entire library. The qualified library constructed is used for cluster preparation and sequencing.

[0050] Bacterial resequencing: (1) After the DNA sample is received, the sample is tested. If the DNA length, total amount and purity meet the requirements of library construction, the subsequent experiments are carried out; (2) DNA samples that pass the electrophoresis test are randomly broken into fragments of about 350bp in length using a Covaris ultrasonic crusher. After the DNA fragments are processed, the library is prepared by end repair, A-tailing, sequencing adapter addition, purification, PCR amplification and other steps; (3) After the library is constructed, Qubit2.0 is used for preliminary quantification, and the library is diluted to 2ng / ffL. Then, the insert size of the library is detected using Agilent 2100. After the fragment meets the expectation, the effective concentration of the library is accurately quantified using the Q-PCR method to ensure the quality of the library; (4) After the library is qualified, different libraries are pooled according to the effective concentration and the target data volume requirements and then sequenced by Illumina Novaseq 6000.

[0051] Figure 3 This is a 16s evolutionary tree based on genomic data. It can be clearly seen that the sequenced strain is closer to Bacillus velezensis.

[0052] Average nucleic acid similarity (ANI) is one of the most powerful measurement indicators for identifying the closeness of bacterial genome relationships. The average value of the comparison of all orthologous protein sequences based on the genome quality inspection reflects the evolutionary distance of the genome quality inspection. When ANI>95%, it means that the two genomes belong to the same species. The results of ANI analysis of the genome and the reference genome are shown in Table 2. The results show that the tested strain is Bacillus velez.

[0053] Table 2. ANI analysis results of genomes and reference genomes

[0054]

[0055] Figure 4 This is the complete genome map of Bacillus Velez L09. The total length of the genome of Bacillus Velez L09 is 3810733bp, with an average GC content of 46.69%. The total number of predicted coding genes is 3751.

[0056] Figure 5 This is the whole genome variation map of Bacillus velez L09. The outermost circle is the reference sequence position coordinates, from the outside to the inside, they are the gene distribution of the reference genome, the InDel distribution of the sample, the number of SNPs, the GC content of the reference sequence genome, and the GC skew value distribution of the reference sequence genome.

[0057] like Figure 6 As shown, three clusters of stimulatory metabolites related to antimicrobial substances were identified in the genome of Bacillus Velez L09, including polyketides, lipopeptides, and bacillusin. The biosynthetic gene clusters of Bacillus Velez and its closely related strains (Bacillus amyloliquefaciens and Bacillus subtilis) have similarities.

[0058] like Figure 7 As shown, a total of 153 CAZymes were detected in the whole genome of Bacillus velezsima L09 strain. CAZymes (153) were divided into five families, such as carbohydrate binding modules (33), carbohydrate lipolytic enzymes (32), glycoside hydrolases (44), glycosyltransferases (40), and polysaccharide lyases (4).

[0059] 5. Preparation of extracellular fluid (CFS), intracellular extract (CFE) and bacterial culture of Bacillus velez L09

[0060] Preparation of extracellular secretion (CFS): Bacillus Velezii L09 strain was cultured in LB medium at 37°C and 180 r / min for 18 h. The cells were collected by centrifugation at 4°C and 6000 r / min for 15 min. The cells were washed three times with sterile 0.1 mol / L phosphate buffer solution, resuspended in PBS, and the bacterial concentration was adjusted to 1×109 The supernatant was filtered through a 0.22 μm water microfiltration membrane to obtain CFS, which was stored at -80°C.

[0061] Preparation of intracellular extract (CFE): Bacillus Velezii L09 was cultured in LB medium at 37°C and 180 r / min for 18 h. The cells were collected by centrifugation at 4°C and 6000 r / min for 15 min. The cells were washed three times with sterile 0.1 mol / L phosphate buffer solution, resuspended in PBS, and the bacterial concentration was adjusted to 1×10 9 CFU / mL. And ultrasonically broken in ice bath conditions. The conditions of ultrasonic breaking are: power 300W, 3s-5s (working 3s, stopping 5s) pulse breaking for 15min. The ultrasonic broken liquid was centrifuged at 4℃ and 12000r / min for 15min. The supernatant was filtered through a 0.22μm water system microfiltration membrane to obtain CFE and stored at -80℃.

[0062] Preparation of bacterial solution: Bacillus Velezii L09 strain was cultured in LB medium at 37°C and 180 r / min for 18 h. The cells were collected by centrifugation at 4°C and 6000 r / min for 15 min. The cells were washed three times with sterile 0.1 mol / L phosphate buffer solution, resuspended in PBS, and the bacterial concentration was adjusted to 1×10 9 CFU / mL.

[0063] VI. In vitro co-culture of Bacillus Velezii L09 against the growth of Aeromonas dakar C160501

[0064] The wild strain of Aeromonas dhaka C160501 was isolated from farmed Siamese crocodiles in Hainan Province, recorded in the literature (Pu, W., G. Guo, N. Yang, Q. Li, F. Yin, P. Wang, J. Zheng, and J. Zeng. "Three Species of Aeromonas (A. Dhakensis, A. Hydrophila and A. Jandaei) Isolated from Freshwater Crocodiles (Crocodylus Sia mensis) with Pneumonia and Septicemia." Lett Appl Microbiol 68, no. 3 (2019): 212 18.), and was isolated and preserved in this laboratory.

[0065] In treatment 1, fresh Bacillus Velezii L09 (20 μL) (1×10 9CFU / mL) were inoculated in 200 mL LB broth and incubated at 37°C for 48 h. In treatment 2, fresh Aeromonas dakarensis C160501 (20 μL) (1×10 9 CFU / mL) were inoculated in 200 mL LB broth and incubated at 37°C for 48 h. In treatment 3, L09 (20 μL) (3 × 105 CFU / mL) and C160501 (20 μL) (1 × 10 9 CFU / mL) were inoculated in 200 mL LB broth and incubated at 37°C for 48 h.

[0066] The samples treated with the above methods were collected at 12h, 24h and 48h. In short, the samples were fixed with 2.5% glutaraldehyde and the fixative was discarded. The samples were rinsed three times with 0.1M, pH7.0 phosphate buffer for 15min each time. The samples were fixed with 1% osmium acid solution for 1-2h. The osmium acid waste solution was carefully removed and the samples were rinsed three times with 0.1M, pH7.0 phosphate buffer for 15min each time. The samples were dehydrated with gradient concentrations of ethanol solution (including 30%, 50%, 70%, 80%, 90% and 95%) for 15min each time, and then treated with 100% ethanol twice for 20min each time. The samples were treated with a mixture of ethanol and isoamyl acetate (V / V=1 / 1) for 30min, and then treated with pure isoamyl acetate for 1h or overnight. Critical point drying.

[0067] like Figure 8 As shown, the morphology of Bacillus velez L09 strain is short and thick, with flat ends and flagella. Most cells are arranged alone and the surface is smooth (Figure A). The morphology of Aeromonas dakar C160501 is long and thin. The two ends are round, with flagella and wrinkles on the surface, and most cells are arranged alone (Figure B). In the 12h co-culture of L09 and C160501, the biomass of C160501 decreased significantly compared with the pure culture of L09 and C160501 (Figure CD). The biomass of C160501 at 48h (Figure F) is further reduced than the biomass of C160501 at 24h (Figure E), and the biomass of C160501 is almost unobservable within the visible range of 48h. It is proved that under pure culture conditions in vitro, Bacillus velez L09 can significantly inhibit the growth of Aeromonas dakar C160501.

[0068] VII. Antagonism of Bacillus velez L09 against Aeromonas dakar C160501 in zebrafish

[0069] During oral infection, 30 zebrafish (average body length 2.5 ± 0.5 cm, average body weight) were divided into three treatment groups, each consisting of 15 fish. All zebrafish were fasted for 12 h before the experiment. In treatment 1, CFS prepared with fresh Bacillus velezensis was used for gavage. In treatment 2, CFS was mixed with Aeromonas dakar C160501 (1 × 10 9 In treatment 3, fresh CFE prepared from Bacillus velez was used for gavage. In treatment 4, CFE and Aeromonas dakar C160501 (1×10 9 In treatment 5, a fresh bacterial suspension (1×10 9 In treatment 6, a fresh bacterial suspension (1×10 9 CFU / mL) and Aeromonas dakar C160501 (1×10 9 CFU / mL)-20 μL gavage. In treatment 7, Aeromonas dakarensis C160501 (1×10 9 CFU / mL)-20 μL was gavage. In treatment eight, PBS-20 μL was gavage.

[0070] 48 h after oral infection, the intestine of each treated zebrafish was fixed with 4% paraformaldehyde. The paraformaldehyde-fixed samples were dehydrated in a graded alcohol series, made transparent with xylene, and then embedded in paraffin. The samples were sectioned to a thickness of 5 μm. After standard hematoxylin and eosin (HE) staining, the tissues were photographed under an Olympus microscope. The results are shown in Fig. 9 The survival rate of zebrafish at 14 days was calculated. Fig.10 .

[0071] Depend on Fig. 9It can be seen that A: The intestinal villi are composed of mucosal epithelium and lamina propria. The mucosal epithelial cells are tall columnar, with oval nuclei and goblet cells. The muscular layer is composed of medial circular muscle and lateral longitudinal muscle, with clear structure and uneven thickness. The serosal layer is composed of a thin layer of connective tissue and a single layer of flat cells. There are no obvious pathological features. B: The mucosal layer of the intestinal tissue folds into the intestinal cavity to form intestinal villi. It can be seen that the intestinal villi are short in length and small in number. The intestinal villi disappear in a small area to form a flat intestinal wall (blue arrow), and the intestinal villi fall off in a small area (black arrow). The muscular layer is exposed, and a small number of intestinal villus epithelial cells have loose and lightly stained cytoplasm (gray arrow), and the number of goblet cells is small. The lamina propria can be seen to have a medium range of loosely arranged connective tissue (green arrow); the muscular layer is uneven in thickness. C: The cytoplasm of many intestinal villus epithelial cells is loose (gray arrows) and irregularly arranged, the number of goblet cells is abundant, and the lamina propria is connective tissue; the muscular layer is composed of medial circular muscle and lateral longitudinal muscle, with clear structure and uneven thickness, and the serosal layer is composed of a thin layer of connective tissue and a single layer of flat cells, all of which have no obvious abnormalities. D: The structure of the intestinal tissue is unclear in a medium range, the intestinal villi are shed in a small range (black arrows), and the lamina propria is exposed; it can be seen that the intestinal villi are short in length and small in number, and the epithelial cells of many intestinal villi are arranged irregularly (gray arrows), and the number of goblet cells is small; the muscular layer structure is clear. E: The mucosal layer of the intestinal tissue folds into the intestinal cavity to form intestinal villi. It can be seen that the intestinal villi are short in length and abundant in number. The intestinal villus epithelial cells fall off in a small area (black arrow), the lamina propria is exposed, the number of goblet cells is small, and the lamina propria can be seen with loosely arranged connective tissue (green arrow). F: The mucosal epithelial cells are tall columnar and arranged neatly. The nucleus is oval and located in the middle or slightly basal part. The number of goblet cells is abundant. The lamina propria is connective tissue, and no obvious abnormalities are found. The muscle layer is composed of the inner circular muscle and the outer longitudinal muscle, with a clear structure and uneven thickness. The serosal layer is composed of a thin layer of connective tissue and a single layer of flat cells, and no obvious abnormalities are found. G: The intestinal villus epithelial cells are arranged closely, the number of goblet cells is small, and multiple foci of loosely arranged connective tissue can be seen in the lamina propria (green arrow). H: The mucosal layer of the intestinal tissue folds into the intestinal cavity to form intestinal villi, which are mostly branched and intertwined. They are composed of mucosal epithelium and lamina propria. The mucosal epithelial cells are tall columnar and arranged neatly. The nuclei are oval and located in the middle or basal part. There are abundant goblet cells. The lamina propria is connective tissue, and a small amount of vascular congestion and dilation can be seen (orange arrows); the muscular layer is composed of the inner circular muscle and the outer longitudinal muscle, with a clear structure and uneven thickness. The serosal layer is composed of a thin layer of connective tissue and a single layer of flat cells. No obvious abnormalities were found.

[0072] In general, compared with the group treated with Aeromonas dakar C160501, co-feeding can alleviate or completely restore to a healthy state to varying degrees. For example, after co-feeding with L09 bacteria and C160501, no obvious abnormalities were found in the pathological sections, and the effect was good.

[0073] Depend on Fig.10It can be seen that after co-feeding, the survival rate of zebrafish infected with Aeromonas dhaka can be improved to varying degrees.

[0074] Obviously, the above embodiments of the present invention are merely examples to more clearly illustrate the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. The crocodile-derived Bacillus Velez strain L09 was deposited in the China Center for Type Culture Collection (CCTCC) on July 31, 2024. The deposit address is Wuhan University, Wuhan, China. The deposit number is CCTCC NO: M 20241703, and the classification name is Bacillus velezensis .

2. The crocodile-derived Bacillus Velez subtilis L09 strain according to claim 1, characterized in that: The total length of the genome of the crocodile-derived Bacillus Velez strain L09 is 3810733 bp, and the average GC content is 46.69%.

3. The crocodile-derived Bacillus Velez subtilis L09 strain according to claim 1, characterized in that: The genome of the crocodile-derived Bacillus Velez strain L09 contains a cluster of stimulating metabolites related to antibacterial substances, including polyketide compounds, lipopeptide compounds, and bacillusin.

4. The crocodile-derived Bacillus Velez subtilis L09 strain according to claim 1, characterized in that: The whole genome of the crocodile-derived Bacillus Velez L09 strain contains five families of CAZymes: carbohydrate binding module, carbohydrate lipolytic enzyme, glycoside hydrolase, glycosyltransferase and polysaccharide lyase.

5. The crocodile-derived Bacillus Velez subtilis L09 strain according to claim 1, characterized in that: The crocodile-derived Bacillus Velezii L09 strain is sensitive to the following antibiotics, including neomycin, nitrofurantoin, streptomycin, chloramphenicol, fosfomycin, tobramycin, roxithromycin, cefazolin, cephalexin, cefoperazone, polymyxin B, cefoxitin, cephradine, ciprofloxacin, sulfamethoxazole, minocycline, lincomycin, co-trimoxazole, clindamycin, spectinomycin, and norfloxacin.

6. An aquatic antibacterial agent, comprising the crocodile-derived Bacillus Velez subtilis L09 strain according to any one of claims 1 to 5.

7. Use of the crocodile-derived Bacillus Velez subtilis L09 strain according to any one of claims 1 to 5 in the preparation of a medicament for antagonizing Aeromonas dakar.

8. The use according to claim 7, characterized in that: The Aeromonas dakariensis is Aeromonas dakariensis C160501.

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